Method for preparative production of long nucleic acids by pcr

ABSTRACT

The invention relates to a method for preparative production of long nucleic acids by PCR. The method involves the following hybridization steps: a) a nucleic acid base sequence is hybridized on the 3′ and 5′ ends with an adapter primer; b) the product from step a) is hybridized on the 3′ and 5′ ends with an extension primer containing an extension sequence, wherein a nucleic acid with extension sequences amplified and enlarged in the 3′ and 5′ ends of the nucleic acid base sequence is then formed from the nucleic acid base sequence. The invention also relates to different applications of the inventive method.

FIELD OF THE INVENTION

The invention relates to a method for preparative production of longnucleic acids by PCR and to different applications for such a method. Asa preparative scale are considered the obtained amounts of nucleic acidswhich are suitable for an immediate application in cell-free proteinbiosynthesis systems and/or in vitro transcription systems. Long nucleicacids are such nucleic acids which contain, in addition to a nucleicacid base sequence (arbitrary length) coding for a protein, furthersequences, in particular regulatory sequences of more than 50, even more70 nucleotides each. Nucleic acids may be DNA or RNA, but also PNA.

BACKGROUND OF THE INVENTION.

Proteins for biotechnological and medical applications are needed with ahigh purity, in particular however also with a high amount, i.e. in themg or g range. In the case of larger proteins, a classical synthesis isnearly impossible and at any case uneconomic.

One possibility to produce proteins on a larger scale is the geneticproduction. For this purpose, cloned DNA coding for the desired proteinis introduced as a foreign DNA in the form of vectors or plasmids, inparticular prokaryotic cells. These cells are then cultivated, wherebythe proteins coded for by the foreign DNA are expressed and obtained. Inthis way considerable amounts of protein may be obtained, however theinsofar known methods, in particular cloning, are expensive.Furthermore, the cells are in most cases only transiently transfected,and in exceptional cases only stably immortalized. A continuousproduction of protein therefore requires a permanent supply of freshcells, which in turn have to be produced by means of the above expensivemeasures.

Another approach is the so-called cell-free in vitro proteinbiosynthesis. Herein, biologically active cell extracts are used, whichare to a high extent freed from naturally occurring cellular nucleicacids and which are reacted with amino acids, energy supplyingsubstances and at least one nucleic acid. The added nucleic acid codesfor the protein to be produced. If DNA is used as the nucleic acid, thepresence of a DNA dependent RNA polymerase is required. Of course, RNA,mRNA may also directly be used. In this way, not only such proteinswhich may also be produced genetically can be produced in a short timeand at comparably moderate expenses, rather such proteins can even beproduced which are for instance cell toxic and consequently cannot beexpressed at all by the usual genetic cell systems at a notable degree.However, it is necessary to produce the added nucleic acid itself, whichagain is expensive by means of genetic methods. In addition, it is oftendesirable to introduce regulatory sequences not naturally linked with aprotein sequence and other sequences, such as spacers, in order toimprove the efficiency of the protein synthesis.

An alternative to the genetic production of complete nucleic acids to beused in the cell-free protein biosynthesis is the so-called expressionPCR. In these connections, the efficient introduction of regulatorysequences (and of other sequences promoting the translation efficiency)into a nucleic acid to be produced plays a special role for theamplification. For the introduction of such further sequences into atarget nucleic acid, very long PCR primers are necessary. Long primersare on the one hand expensive to produce and increase on the other handthe probability of the generation of inhomogeneous PCR products.

PRIOR ART

From the document U.S. Pat. No. 5,571,690 it is known in the art toproduce a nucleic acid in a preparative scale by means of PCR, whereinthe nucleic acid to be amplified already contains all necessaryregulatory sequences. The insofar known measures do therefore not permitan introduction of other, better regulatory sequences or a replacementof existing regulatory sequences by such other better sequences.Further, the nucleic acid obtained from the amplification cannotimmediately be used in the protein synthesis. Finally, a specificnucleic acid from a nucleic acid mixture cannot be amplified, with asimultaneous conversion of the target gene for the protein biosynthesiscoded by the specific nucleic acid.

From the document WO-A-9207949 it is known in the art to produce andamplify in several steps a nucleic acid with a sequence coding for aprotein and with a regulatory sequence. In a first step, the sequencecoding for the protein is amplified with a standard PCR. An upstreamhybridizing primer serves for the introduction of an adapter sequencefor a so-called “overlap extension PCR”. In a parallel second step thehybridization partner for the overlap extension PCR is prepared. Forthis purpose, two partially complementary primers are hybridized andfilled up. The obtained product carries at the 3′ end the sequence ofthe adapter sequence being homologous to the 5′ end of the amplicon ofthe first step as well as a promoter sequence and regulation elementsfor the cell-free protein biosynthesis. The third step finally is theoverlap and extension reaction. The products of the first two steps arehybridized, filled up so to form a double strand and finally amplifiedwith further primers. In this final amplification, an additionalsequence is incorporated via a primer ahead of the promoter, with thepurpose of an improved transcription. In two further steps, thetranscription as well as the translation in a cell-free system takesplace. It is disadvantageous, herein, that in total four steps arenecessary for obtaining the desired mRNA. Further, the 3′ sequencesbeing necessary for the protein biosynthesis in prokaryotic systems arelacking. Further, no affinity tag sequences or the like are introduced,by means of such sequences the purification of the obtained proteinbeing facilitated. Due to the complexity of the method and the lack of3′ sequences for prokaryotic systems, the insofar known method shouldneither be useful for prokaryotic systems nor for an application tonucleic acid mixtures (cDNA or genome libraries).

In the document Martemyanov, K. A., et al.; FEBS Lett. 414:268-270(1997), a method is described being similar to that of the documentWO-A-9207949. There are differences in that a sequence homology betweenthe 5′ end of the upstream and the 3′ end of the downstream primerexists. Thereby a multimerization takes place, and at long last apolyprotein is produced which is then again cleft into monomers. Inaddition it is disadvantageous, in this variant, that polyproteins ofvery few proteins only can chemically be cleft. Furthermore, the yieldis relatively low.

From the document Nakano, H., et al.; Biotechnol. Bioeng. 64:194-199(1999), a special protein bioreactor for the expression of PCR productsin Escherichia coli lysate is known in the art. Therein, standard PCRproducts are used without special methods for the generation thereof.The PCR product is expressed in a distinctly poorer condition than in aplasmid.

All above documents relate to eukaryotic systems.

TECHNICAL OBJECT OF THE INVENTION

The invention is based on the technical object to provide a method forthe production of long nucleic acids, in particular with proteinsequences as well as with selected regulatory sequences, wherein saidmethod needs low expenditure, generates high amounts of product nucleicacids, is suited without additional expenditure for prokaryotic systems,and by means of which an amplification of defined nucleic acids fromnucleic acid libraries is possible.

BASICS OF THE INVENTION.

For achieving the above technical object, the invention teaches a methodfor preparative production of long nucleic acids by means of PCR andinvolving the following hybridization steps: a) a nucleic acid basesequence is hybridized on the 3′ and 5′ ends with an adapter primer, b)the product from step a) is hybridized on the 3′ and 5′ ends with anextension primer containing an extension sequence, wherein a nucleicacid enlarged by the extension sequences and amplified on the 3′ and 5′ends of the nucleic acid base sequence is then formed from the nucleicacid base sequence.

A nucleic acid base sequence is a sequence coding for a protein. Inparticular this may be a gene, however also sequences from intronlessgenomes. The extension sequences may in particular be sequencescomprising a regulatory sequence and/or sequences comprising a ribosomalbinding sequence. The adapter primers are comparatively short. One partof an adapter sequence is specific for the nucleic acid base sequence,another part is constant and hybridizes an extension sequence.

This means that it is not necessary to use respectively “fitting” longextension sequences for different nucleic acid base sequences. Rather,the comparatively short adapter primers only have to be adjusted to adefined nucleic acid base sequence, whereas the extension sequences mayso to speak be universal, i.e. for different nucleic acid base sequencescan always be used the same or several few selected extension sequences.Thus the extension sequences produced in an expensive way can be put toa wider use, and for a specific nucleic acid base sequence only theadapter sequences need to be produced. This is however little expensive,since the adapter sequences can be relatively short.

This permits for instance to add a regulatory sequence as well as aribosomal binding sequence, each via one of the extension primers, to anucleic acid base sequence, and that even in one PCR step. Thus anucleic acid can be obtained which results in a particularly hightranscription and/or translation efficiency in a prokaryotic system ofthe cell-free protein biosynthesis.

A particular advantage of the method according to the invention is thatit is a generally applicable method for arbitrary coding sequences.

The hybridization with a primer on the 3′ and 5′ ends relates inparticular to double-stranded nucleic acids, the hybridization of theprimer respectively occurring on the 5′ end of the sense and antisensestrands. Referred to the single strand, the hybridization with thevarious primers described above and below takes place on the respective5′ end.

EMBODIMENTS OF THE INVENTION

Of an independent importance is a embodiment of the invention, whereinthe product from step b) can be hybridized in a step c) on the 3′ and 5′ends with respectively one amplification primer, and an amplifiednucleic acid end sequence is formed. The amplification primers, too, areon the one hand relatively short und universally applicable, andconsequently easily available. By means of the amplification primers,moreover further (shorter) sequences can be added on the ends, suchsequences further increasing the translation efficiency. By means of theshort amplification primers, variations and modifications on the ends ofthe nucleic acids can also easily be introduced. This is in particularadvantageous, since thus for variations and modifications no differentextension primers need to be produced, which again would be expensive ina disturbing manner.

An example for a variation or modification is the incorporation of abiotin residue, coupled on the 5′ end of the amplification primer.Hereby is obtained, after incubation of the nucleic acid end sequencewith for instance biotin-binding streptavidin, a nucleic acid endsequence being stable against exonuclease disintegration which shows anincrease in the half-life period in an in vitro protein biosynthesissystem compared to a not stabilized nucleic acid end sequence by amultiple, typically more than 5 times, for instance from approx. 15 minto approx. 2 h. There are obtained stabilities which are comparable tothose of circular plasmids and thus can replace them in an equivalentmanner. An alternative is a stabilization by means of digoxigeninbinding anti-digoxigenin antibodies. The stabilizing group may beprovided on both ends of the nucleic acid end sequence. Another exampleis a modification with an affinity tag or a sequence coding therefor oran anchor group or a sequence coding therefor. A anchor group permits animmobilization by binding the anchor group to a solid body surface withmatched binding sites. The anchor group may be disposed at the nucleicacid itself, however a sequence coding therefor may also be provided.

The adapter primers typically contain <70, in particular 20 to 60nucleotides. The extension primers typically contain ≧70, even 90 andmore nucleotides. The amplification primers finally typically contain<70, in most cases <30 nucleotides, typically >9 nucleotides. Just theadapter primers need to be specifically adjusted to a defined nucleicacid base sequence, which requires little efforts because of therelatively short sequences.

Advantageously, the steps a), b) and as an option the step c) areperformed in a PCR solution containing the nucleic acid base sequence,the adapter primers, the extension primers and as an option theamplification primers. This is then a one-step PCR with in total sixprimers, two adapter sequences, two extension sequences and twoamplification sequences. It is sufficient to use the adapter primers andthe extension primers at low concentrations and to thus generate aninsofar low amount of intermediate product. The intermediate productneeds not to be present in a homogeneous form, thus expensiveoptimizations are not required. Because of the shortness of theamplification primers, even for the amplification to the high amount ofnucleic acid end sequences, no optimizations are needed.

Alternatively to the above embodiment has an independent importance avariant comprising two PCR steps. Therein, the steps a) and b) in amethod step A) are performed in a pre-PCR solution containing thenucleic acid base sequence, the adapter primers and the extensionprimers for a defined first number of cycles, and the step c) in amethod step B) is performed in a main PCR solution containing the PCRproduct from the step A) and the amplification primers for a definedsecond number of cycles. Step A) may be performed in a reaction volumebeing ½ to 1/10 of the reaction volume of step B). In step A) will thenbe generated, due to the lower volume, a higher concentration ofintermediate product, or a distinctly lower amount of nucleic acid basesequence can be used. By the dilution by means of PCR starting volume atthe transition from step A) to step B), in turn the adapter primes andthe extension primers are substantially diluted with the consequence ofan increase in the probability of the incorporation of variations and/ormodifications in the nucleic acid end sequences via the amplificationprimers.

In detail, the procedure in the first above alternative may be that thePCR is performed in a reaction volume of 10 to 100 μl, preferably 20 to40 μl with 0.01 to 100 pg, preferably 1 to 50 pg nucleic acid basesequence, 0.05 to 10 μM, preferably 0.1 to 5 μM adapter primer, and0.005 to 0.5 μM, preferably 0.001 to 0.1 μM extension primer, whereinafter a defined number of starting cycles 0.01 to 10 μM, preferably 0.1to 10 μM amplification primer are added, and wherein by means of adefined number of subsequent cycles the amplified nucleic acid endsequence is produced. In the second above alternative, the followingreaction conditions are recommended: step A): reaction volume <10 μl;0.001 to 5 pg, preferably 0.01 to 1 pg nucleic acid base sequence; 0.05to 10 μM, preferably 0.1 to 5 μM adapter primer, and 0.05 to 10 μM,preferably 0.1 to 5 μM extension primer; first number of cycles 10 to30, preferably 15 to 25, step B): reaction volume 10 to 100 μl,preferably 15 to 50 μl, obtained by complementing the solution from stepA) with PCR starting solution; 0.01 to 10 μM, preferably 0.1 to 5 μMamplification primer; second number of cycles 15 to 50, preferably 20 to40.

The invention further teaches the use of the method according to theinvention for the production of nucleic acids for the cell-free in vitroprotein biosynthesis, in particular in prokaryotic systems, preferablyin a translation system of Escherichia coli D10.

A method according to the invention can advantageously be used for theselective amplification of a defined nucleic acid base sequence from anucleic acid library. This permits a characterization of gene sequences,wherein the gene sequence is used as a nucleic acid base sequence andwherein the obtained protein is analyzed with regard to structure and/orfunction. The background of this aspect of the invention is that formany genes the sequences are known, not however the structure andfunction of the protein coded thereby. Thus elements of a gene libraryof which only the sequence as such is known, can be examined for theirfunction in an organism. The examination of the structure and functionof the obtained protein follows the conventional working methods of thebiochemistry.

By the method according to the invention nucleic acids may be obtained,which contain a nucleic acid base sequence coding for the protein and aribosomal binding sequence and as an option one or several sequences ofthe group comprising “promoter sequence, transcription terminatorsequence, expression enhancer sequence, stabilization sequence andaffinity tag sequence”. An affinity tag sequence codes for a structurehaving a high affinity for (in most cases immobilized) binding sites inseparation systems for the purification. Thus an easy and highlyaffinitive separation of proteins not containing the affinity tag ispossible. An example therefor is Strep-tag II, a peptide structure of 8amino acid residues with affinity to StrepTactin. A stabilizationsequence codes for a structure which either itself or after binding to aspecific binding molecule being specific for the structure causes astabilization against degradation, in particular by nucleases. Astabilization of a nucleic acid (end) sequence may also take place bythat on one end, preferably on both ends, a biotin group is incorporatedwhich can be reacted with streptavidin. This incorporation may beeffected by using primers carrying biotin, in particular amplificationprimers. An expression enhancer sequence increases the translationefficiency compared to a nucleic acid without expression enhancersequence. For instance (non-translated) spacers may be used for thispurpose. A transcription terminator sequence terminates the RNAsynthesis. An example is the T7 phage gene 10 transcription terminator.Transcription terminator sequences can also stabilize againstdegradation by 3′ exonucleases. Advantageous relative arrangements ofthe above sequence elements with respect to each other can begeneralized from the following examples of execution.

In the following, the invention is explained in more detail, based onexamples representing preferred embodiments only.

Methods:

PCR:

The PCR was performed in a reaction volume quantified in the exampleswith 10 mM Tris-HCl (pH 8.85 at 20° C.), 25 mM KCl, 5 mM (NH₄)₂SO₄, 2 mMMgSO₄, 0.25 of every dNTP, 3 U Pwo DNA polymerase (Roche) and the amountof nucleic acid base sequence specified in the examples. The cycles wereperformed for 0.5 min at 94° C., 1 min at 55° C. and 1 min at 72° C.

In vitro expression:

In vitro experiments were made according to the document Zubay, G.;Annu. Rev. Genet. 7:267-287 (1973) with the following modifications. TheEscherichia coli S-30 lysate was supplemented with 750 U/ml T7 phagesRNA polymerase (Stratagene) and 300 μM [¹⁴C] Leu (15 dpm/pmol,Amersham). PCR products and control plasmids were used in concentrationsof 1 nM to 15 nM. The reactions were performed at 37° C., the coursebeing monitored by that at subsequent times 5 μl aliquots were takenfrom the reaction mixture and the incorporation of [¹⁴C]Leu wasestimated by means of TCA precipitation. Further 10 μl aliquots weretaken for the purpose of an analysis of the synthesized protein by meansof SDS-PAGE, followed by an autoradiography in a phosphoimager system(Molecular Dynamics).

Plasmid Construction:

A high copy derivative of the plasmid pET BH-FABP (Specht, B. et al.; J.Biotechnol. 33:259-269 (1994)) coded for bovine heart fatty acid bindingprotein, called pHMFA, was constructed. A fragment of pET BH-FABP wasproduced by digestion with the endonucleases SphI and EcoRI and insertedinto the vector pUC18. With regard to the sequences being relevant forthe synthesis of H-FABP, the plasmid pHMFA is identical with theoriginal plasmid. It should be noted that the linearized plasmid doesnot behave in a better way than the circular plasmid.

Construction of Nucleic Acids With Different Sequence Regions Upstreamof the Promoter:

The plasmid pHMFA served as a matrix for the construction of nucleicacids with different sequence regions upstream of the promoter. Theconstructs (see examples) FA1, FA2 and FA4 with 0, 5 and 249 base pairsupstream of the promoter were generated with the primers P1, C1 and P2as well as with the downstream primer P3. The construct FA3 with asequence region of 15 base pairs upstream of the promoter was obtainedby digestion of FA4 with the endonuclease Bgl II. The control plasmidpHMFA(EcoRV) with a sequence region of 3.040 base pairs was obtained bydigestion of the plasmid with EcoRV. All products were purified byagarose gel electrophoresis, followed by gel extraction by means of the“High Pure PCR Product Purification Kit”.

Affinity Purification:

The purification of the fatty acid binding protein containing Strep-tagII (Voss, S. et al.; Protein Eng. 10:975-982 (1997)) was performed bymeans of affinity chromatography according to manufacturer'sinstructions (IBA Goettingen, Germany), with the deviation of a reducedvolume of the affinity column (200 μl). The reaction mixture of thecoupled transcription/translation was centrifuged for a short time andthen applied to the column. Isolated fractions were analyzed by TCAprecipitation and audioradiography after SDS-PAGE (see above).

H-FABP Activity Assay:

The complete reaction mixture with H-FABP synthesized in vitro wasexamined for the activity of the binding of oleic acid. Various volumes(0 to 30 μg) were filled up to 30 μl with reaction solution withoutH-FABP and diluted with translation buffer (50 mM HEPES pH 7.6, 70 mMKOAc, 30 mM NH₄Cl, 10 mM MgCl₂, 0.1 mM EDTA, 0.002% NaN₃ so to obtain anend volume of 120 μl. After addition of 2 μl 5 mM [9,10(n)-³H] oleicacid (Amersham) with a specific activity of 1,000 dpm/pmol, the sampleswere incubated for one hour at 37° C. 50 μl of the samples were used forremoval of unbound oleic acid by means of gel filtration (Micro Bio-SpinChromatography columns; Bio-Rad). The ³H radioactivity of the elutedfractions was measured by means of a scintillation counter.

Analysis of the Stability of the Nucleic Acids:

Radioactively marked nucleic acids were synthesized according to theabove conditions, however in presence of 0.167 μCi/μl [α-³⁵S] dCTP. Themarked nucleic acids were used in a coupled transcription/translation,reaction volume 400 μl. 30 μl aliquots were taken at subsequent times.After addition of 15 μg ribonuclease A (DNAse-free, Roche), they wereincubated for 15 min at 37° C. Another incubation for 30 min at 37° C.was performed after addition of 0.5% SDS, 20 mM EDTA and 500 μg/mlproteinase K (Gibco BRL) in a total reaction volume of 60 μl. Theremaining PCR products were further purified by means of ethanolprecipitation and then subjected to a denaturating electrophoresis (5.3%polyacrylamide, 7 M urea, 0.1% SDS, TBE). The dried gel was passed forthe quantification of the radioactivity through a phosphoimager system(Molecular Dynamics).

Sequences:

The employed primer sequences are shown in FIG. 1.

EXAMPLE 1

PCR with Four Primers.

In FIG. 2 is shown a diagrammatic representation of a one-step PCRaccording to the invention with four primers. In the center, the nucleicacid base sequence coding for a protein can be seen, said nucleic acidbase sequence comprising the complete coding sequence for H-FABP(homogeneous and functionally active fatty acid binding protein frombovine heart), obtained as a 548 bp restriction fragment from PHMFA bydigestion by means of the endonucleases Ncol and BamHI (and a 150 bpsequence on the 3′ end which is neither translated nor complementary toan adapter primer or extension primer). Thereto the two adapter primersA and B are hybridized, which have ends being homologous with the endsof the nucleic acid base sequence. The adapter primer A further containsa ribosomal binding sequence. To the outside ends of the adapter primersA and B are hybridized the extension primers C and D. The extensionprimer C comprises the T7 gene 10 leader sequence including the T7transcription promoter as wall as upstream a sequence of for instance 5nucleotides. The extension primer D comprises the T7 gene 10 terminatorsequence.

EXAMPLE 2

Efficiency of the H-FABP Synthesis in Dependence from the SequenceRegion Upstream of the Promoter.

Four PCR products (FA1 to FA4) with different sequence regions upstreamof the promoter (0, 5, 15, 250 base pairs) and the linearized controlplasmid pHMFA(EcoRV) with 3,040 bp upstream of the promoter wereexamined in different concentrations (1, 5, 10 and 15 mM) for in vitrotranscription/translation. FIG. 3 shows that all sequence regions except0 (lower curve) cause an increase in the protein synthesis. 5 base pairsalready are sufficient.

EXAMPLE 3

Improvement of the H-FABP Synthesis by the Phage T7 Gene 10Transcription Terminator/5′ Leader Sequence Phage T7 Gene 10.

In FIG. 4 can be seen that by the phage T7 gene 10 transcriptionterminator the synthesis can be at least improved by factor 2.8. Thetriangles are for FAΔt, and the squares for FAt (see also FIG. 2).

Further, it can be seen from FIG. 4 that by the a deletion of 34 bpbetween the beginning of transcription and the epsilon sequence (Olins,P. O. et al.; Escherichia coli, J. Biol. Chem. 264:16973-16976 (1989))leads to a suppression of a product generation. The circles are for thisvariant FAA34 (see also FIG. 2).

EXAMPLE 4

Influence of the Position of the Transcription Terminator Sequence.

For examining the influence of the position of the terminator sequence,the products FAst and FAast were produced (see FIG. 2). Both areidentical with FAt and FAat, with the exception that a 22 bp spacersequence is introduced between the stopcodon and the terminator by meansof different primers. In FIG. 5 can be seen that the spacer sequencecauses an approx. 2-fold increase in the expression.

Further, it can also be seen in FIG. 5 from a comparison of FAt and FAatthat an affinity tag has nearly no influence on the expression.

EXAMPLE 5

PCR from a Complex DNA Mixture.

The effectivity and specificity of the method according to the inventionwas examined in presence of a high amount of competitive DNA. A PCR forFAst was performed according to the above descriptions, with thefollowing exceptions: the nucleic acid base sequence was performed inconcentrations from 0.16 to 20 pg/50 μl reactor volume, and thereactions were supplemented with 0.83 μg chromosomal DNA fromEscherichia coli, ultrasonically treated for 5 min. It was found thatneither the quality nor the quantity of the PCR product was influencedby the presence of a 5 million-fold excess of competitive DNA.

EXAMPLE 6

Affinity Purification with Strep-Tag II.

A reaction mixture of 10 μg of the radioactively marked FAast wassubjected to the affinity purification. Approx. 81% of the appliedmaterial were obtained from the column, and 67% could be gained as apure product in the elution fractions (calculated from TCA precipitationof the fractions of the affinity column).

EXAMPLE 7

Activity of the PCR Product.

Samples of H-FABP, synthesized either by means of the plasmid or as PCRproduct FAast were examined together with regard to the binding activityfor oleic acid. After the transcription/translation, various volumes of0 to 330 pmol of non-marked H-FABP were examined in a binding assayaccording to the above description of methods. The activities were foundas being identical, irrespective of the way of production.

EXAMPLE 8

Stability of the PCR Product.

For examining whether the stability of the PCR product would possiblylimit the effectivity of the expression, the decrease of the PCR productFAast was measured. For this purpose, the radioactively marked productwas used. In certain time intervals, aliquots of the reaction mixturewere taken and examined by means of denaturated polyacrylamide gelelectrophoresis. The amount of remaining PCR product was quantified byscanning the radioactivity of the gel and compared to the time course ofthe protein synthesis, measured by scanning the radioactivity of H-FABPin the gel after separation of the reaction mixtures by means ofSDS-PHAGE. The results are shown in FIG. 6. It can be seen that thehalf-life period of the PCR product is approx. 100 min, whichcorresponds to the time when the H-FABP synthesis reaches a horizontalline.

EXAMPLE 9

Optimized Conditions for a PCR with Four Primers.

In Table I are summarized optimized conditions for a PCR with fourprimers in a reaction volume of 25 μl. TABLE I a) Reaction componentsConcentration in Reaction component the reaction PCR buffer for Pwopolymerase (Roche) acc. to supplier Desoxynucleotidetriphosphates dATP,dCTP, dGTP 0.25 mM and dTTP Adapter primer a (55 nucleotides) 0.1 μMAdapter primer b (51 nucleotides) 0.1 μM Extension primer c (75nucleotides) 0.4 μM Extension primer d (95 nucleotides) 0.4 μM Template:coding sequence for fatty acid 10 pg/25 μl binding protein/restrictionfragment from pHM18FA (Ncol/BamHI) Pwo DNA polymerase (Roche) 1.5 U/25μl b) Temperature program Temperature cycle Segment 1 30 sec 94° C.Segment 2 60 sec 55° C. Segment 3 60 sec 72° C. 60 cycle repetitions

EXAMPLE 10

PCR with Six Primers.

With the materials from example 9, however with two additionalamplification primers e (26 nucleotides) and f (33 nucleotides) and anincreased adapter primer concentration of 0.2 μM, varying extensionprimer concentrations were adjusted. With regard to the amplificationprimers, reference is made to FIG. 1, BIOR and BIOF. BIOF is abiotin-marked forward primer and BIOR a biotin-marked reverse primer.The structure is shown in FIG. 7.

A minimum demand of expensive extension primers resulted, if first 25cycles without amplification primer and then another 25 cycles withamplification primer were performed. The concentration of the extensionprimer could be reduced by the use of the amplification primers down to0.025 μM, a factor of approx. 1/20, with nevertheless improvedhomogeneity and yield of PCR product.

These advantages are based on that the probability of the generation ofintermediate products in high concentrations is strongly reduced withthe use of the six primers, since the primers required for thegeneration of the intermediate products are used in low concentrations.Intermediate products thus cannot be exponentially enriched with theamplification primers.

EXAMPLE 11

PCR with Six Primers in Two Steps.

In principle, the materials are used as described above. First, apre-PCR is performed in a reaction volume of 5 μl with 0.1 μg nucleicacid base sequence, with 0.3 μM adapter primer and 0.5 μM extensionprimer over 20 cycles. Then the reaction solution thus obtained isdiluted with PCR starting volume to 25 μl. Then amplification primer isadded to an end concentration of 0.5 μM. Finally, it is amplified foranother 30 cycles.

EXAMPLE 12:

Stabilization of a Nucleic Acid with Biotin.

By using the primers BIOF and BIOR in a PCR with 6 primers, as describedabove, a nucleic acid was produced, and the decomposition thereof as afunction of the time and the improvement of the protein synthesis wereexamined. This is shown in FIGS. 7 and 8. It can be seen that withbiotin, in particular after reaction with streptavidin, a substantiallybetter stability is obtained. This also leads to a protein synthesisbeing higher up to 20%.

Irrespective of the above examples, it has to be noted that with themethod according to the invention, variations of the sequences are alsovery easily possible by mutations, for instance by using Taq polymeraseand/or modified reaction conditions. If this is not desired, preferablyPwo or Pfu can be used which function in a more precise manner and haveproof-reading activity.

1. A method for preparative production of long nucleic acids by means ofPCR and involving the following hybridization steps: a) a nucleic acidbase sequence is hybridized on the 3′ and 5′ ends with an adapterprimer, b) the product from step a) is hybridized on the 3′ and 5′ endswith an extension primer containing an extension sequence, wherein anucleic acid sequence enlarged by the extension sequences and amplifiedon the 3′ and 5′ ends of the nucleic acid base sequence is formed fromthe nucleic acid base sequence.
 2. A method according to claim 1,wherein the product from step b) is hybridized in a step c) on the 3′and 5′ ends with one amplification primer each, an amplified nucleicacid end sequence being formed.
 3. A method according to claim 1 or 2,wherein the adapter primers contain <70 nucleotides, wherein theextension primers contain ≧70 nucleotides, and/or wherein theamplification primers contain <70 nucleotides.
 4. A method according toone of claims 1 to 3, wherein the steps a), b) and as an option the stepc) are performed in a PCR solution containing the nucleic acid basesequence, the adapter primers, the extension primers and as an optionthe amplification primers.
 5. A method according to one of claims 1 to4, wherein the steps a) and b) in a method step A) are performed in apre-PCR solution containing the nucleic acid base sequence, the adapterprimers and the extension primers for a defined first number of cycles,and wherein the step c) in a method step B) is performed in a main PCRsolution containing the PCR product from the step A) and theamplification primers for a defined second number of cycles.
 6. A methodaccording to one of claims 1 to 4, wherein the PCR is performed in areaction volume of 10 to 100 μl, preferably 20 to 40 μl with 0.01 to 100pg, preferably 1 to 50 pg nucleic acid base sequence, 0.05 to 10 μM,preferably 0.1 to 5 μM adapter primer, and 0.005 to 0.5 μM, preferably0.001 to 0.1 μM extension primer, wherein after a defined number ofstarting cycles 0.01 to 10 μM, preferably 0.1 to 5 μM amplificationprimer are added, and wherein by means of a defined number of subsequentcycles the amplified nucleic acid end sequence is produced.
 7. A methodaccording to claim 5, comprising the following reaction conditions: stepA): reaction volume <10 μl; 0.001 to 5 pg, preferably 0.01 to 1 pgnucleic acid base sequence; 0.05 to 10 μM, preferably 0.1 to 5 μMadapter primer, and 0.05 to 10 μM, preferably 0.1 to 5 μM extensionprimer; first number of cycles 10 to 30, preferably 15 to 25, step B):reaction volume 10 to 100 μl, preferably 15 to 50 μl, obtained bycomplementing the solution from step A) with PCR starting solution; 0.01to 10 μM, preferably 0.1 to 5 μM amplification primer; second number ofcycles 15 to 50, preferably 20 to
 40. 8. The use of a method accordingto one of claims 1 to 7 for the production of nucleic acids for thecell-free in vitro protein biosynthesis, in particular in prokaryoticsystems, or for in vitro transcription systems.
 9. The use according toclaim 8 in a translation system of Escherichia coli D10.
 10. The use ofa method according to one of claims 1 to 7 for the selectiveamplification of a defined nucleic acid base sequence from a nucleicacid library.
 11. The use of a method according to one of claims 1 to 7for the characterization of gene sequences, wherein the gene sequence isused as a nucleic acid base sequence and wherein the obtained protein isanalyzed with regard to structure and/or function.
 12. A nucleic acidfor cell-free protein biosynthesis systems which contains a basesequence coding for a protein and a ribosomal binding sequence as wellas an option one or several sequences of the group comprising “promotersequence, transcription terminator sequence, expression enhancersequence, stabilization sequence and affinity tag sequence”.